[Construction, eukaryotic expression and biological activities of a recombinant human single chain interleukin-12...

To explore the feasibility of recombinant human interleukin 12 (IL-12) with biological activities by molecular biological techniques.

Both p40 and p35 subunits cDNA of human IL-12 were cloned from mRNA extracted from NC-37 cell line by using RT-PCR, and the fusion gene (p40-linker-p35) of recombinant human single chain IL-12 (rhscIL-12) was constructed by using a polypeptide linker (Gly(4)Ser)(3). rhscIL-12 eukaryotic expressing vector pcDNA3.1 (+)-hscIL-12 was constructed by inserting the rhscIL-12 fusion gene into pcDNA3.1 (+) eukaryotic expressing plasmid. COS-7 cells were transfected with pcDNA3.1 (+)-hscIL-12 plasmid.

A stable rhscIL-12 expressing cell line COS-rhscIL-12 was obtained by G418 selection. Western blot showed the presence of a 70 x 10(3) band of the fusion protein, which specifically bond to mouse-anti-human IL-12 monoclonal antibody. The assays of biological functions showed that the fusion protein had strong bioactivities in stimulating the lymphocyte proliferation, enhancing the NK cell cytotoxicity and increasing the IFN-gamma production.

The constructed rhscIL-12 fusion gene could express biologically functional rhscIL-2 fusion protein.